1. Field of the Invention
The present invention relates to nucleic acid molecules, comprising a nucleotide sequence encoding a polypeptide with chalcone 3-hydroxylase activity.
2. Description of Related Art
The flower color is one of the most striking characteristics of ornamental plants and is therefore a significant factor for their market value. Beside traditional growing, genetic approaches for the creation of new species increasingly gain significance. Examples for that are the generation of blue carnations as well as that of the so-called blue rose, which is already commercially available in a great many countries worldwide.
The development of the flower colors is mainly based on the presence of two different pigment groups, the carotenoids and the flavonoids. The flavonoid class of anthocyanins is mainly responsible for the formation of the red, blue and purple flower colors, while the yellow flower color in most of the cases results from the accumulation of carotenoids. In some plant species, however, the yellow plant color is formed by yellow flavonoids and their biosynthetically related anthochlor pigments (chalcones and aurones). Therefore, a modification of the chalcone or flavonoid metabolism, respectively, may decisively contribute to the alteration of the flower colors. Beside growing plants with blue flowers, the introduction of the yellow flower color in ornamental plants, of which no or only occasional yellow varieties are available, is of particular interest. Often only small modifications in the pigment structure result in drastic color changes. This above all applies to the number of the hydroxyl groups in the basic structures.
Although many flavonoids in their chemically pure form have a pale yellow color, their presence in petals does not result in the development of the yellow flower color. Beside the common 5,7-hydroxylation pattern of the A-ring, the so-called “yellow flavonols” have an additional hydroxyl group at positions 6 or 8, which effects an absorption shift into the longer-wave region and thus an intensification of the yellow color. The presence of such higher hydroxylated compounds results in the development of the yellow flower color. Quercetagetin was first identified as the yellow pigment in various species of marigold and is also present in the flowers of Rudbeckia hirta. 
Yellow flavones represent the main pigments in some flowers of Asteraceae. While the common prevalent flavones do not result in the development of yellow flower colors, the presence of an additional hydroxyl group at position 2′ of the B-ring of luteolin effects a yellow coloration of the pigment. Isoetin (2′-hydroxyluteolin) was identified as the yellow main pigment of Heywoodiella oligocephala. The introduction of a hydroxylase, which catalyses the 2′-hydroxylation, could result in the formation of yellow-colored flavones in the flowers of transgenic plants, which generally only produce common flavones.
Contrary to the yellow flavonols, for which the presence of an additional hydroxyl group results in the development of the yellow flower color, the loss of a hydroxyl group at position 3 of anthocyanins or anthocyanidins, respectively, is responsible for a shift of the absorption into the shorter-wave region and thus for the orange and yellow color of the so-called 3-deoxyanthocyanins. 3-deoxyanthocyanins are rare plant pigments, which as such exist in only a few plants like Gesneriaceae, Zea mays (maize) and in species of Sorghum (millet). Three representatives of this group could be identified, apigeninidin (3-deoxypelargonidin), luteolinidin (3-deoxycyanidin) and columnidin. Of these, however, only apigeninidin derivatives contribute to the yellow flower color. The others have an orange to light red coloration. The biochemical formation of the flavan-4-ols as precursors for the 3-deoxyanthocyanins is caused by the reduction of the carbonyl group of the flavanones at position 4. This reaction is catalysed by dihydroflavonol-4-reductase in a high number of cultivated and ornamental plants, however, commonly only takes place in plants, in which the FHT reaction is inhibited.
The deep yellow anthochlor pigments (chalcones and aurones) have only a limited spread in nature, however, frequently exist in species of Asteraceae or Scrophulariaceae. In general, two types of chalcones can be synthesised in the flowers, the 6′-hydroxychalcones (phloroglucinol type) and the 6′-deoxychalcones (resorcinol type). The respective aurones are the 4-hydroxy- and the 4-deoxyaurones (identical position, different numbering of the rings). 6′-deoxychalcones are formed by chalcone synthase together with chalcone ketide reductase (CHKR, synonyms polyketide reductase, PKR, chalcone reductase) via a polyketide intermediate.
Chalcones are secondary plant metabolites and biochemical precursors for all flavonoid classes. Therefore, and due to their physiological functions in plants, like e.g. the influence on the flower color, they play an important role in the plant physiology. Beside the common 6′-hydroxychalcones, which represent intermediates of the biosynthesis of the widespread 5-hydroxyflavonoids, the more rare 6′-deoxychalcones are often accumulated in the flowers of Asteraceae species, since chemically they cannot be converted into the respective 5-deoxyflavanones and are also not accepted as substrates by the chalcone isomerases (CHIs) of most plants. The accumulation of 6′-deoxychalcones results in the development of the yellow flower color. 6′-hydroxychalcones, on the other hand, are accumulated in the plant tissue in rare cases only, since they can be easily converted into flavanones enzymatically or chemically. Therefore, in a few cases only, they are responsible for the yellow coloration of flowers, as in the yellow flowers of carnations (Dianthus caryophyllus), snapdragon (Antirrhinum majus) and everlasting flowers (Helichrysum bracteatum), since beside the CHI, these mutants are lacking at least one more enzymatic activity of the flavonoid metabolism.
Like flavonoids, chalcones, too, can have further hydroxyl groups beside the hydroxyl group at position 4 (corresponding to position 4′ in flavonoids) in the B-ring, namely at positions 3 or 3 and 5 (corresponding to positions 3′ or 3′ and 5′ in flavonoids). Contrary to the very well investigated hydroxylation of flavonoids at positions 3′ and 3′, 5′, which are catalysed by the cytochrome P450-dependent monooxygenases flavonoid-3′-hydroxylase (F3′H) or flavonoid-3′,5′-hydroxylase (F3′, 5′H), respectively, for a long time there has been uncertainty about which enzyme is responsible for the introduction of additional hydroxyl groups in the B-ring of chalcones. It could be demonstrated that the introduction of a hydroxyl group at position 3 of 6′-deoxychalcones is catalysed by a cytochrome-P450-dependent monooxygenase. Investigations with recombinant F3′Hs of various plants, which accumulate chalcones in their petals, as well as of such ones, which do not accumulate chalcones, demonstrated, however, that these F3′Hs are not able to catalyse the hydroxylation of chalcones.
As mentioned already, the F3′Hs are membrane-bound cytochrome (cyt) P450-dependent monooxygenases. The super family of cyt P450 enzymes is a group of very different enzymes, which catalyses many different and complex oxygenation reactions with a high number of substrates in the presence of NADPH or NADH. They include a haem group and exist in prokaryotes as well as in eukaryotes. In plants, an extraordinarily high number of cyt P450 genes can be found. In Arabidopsis for example, 272 cyt P450 genes could be detected. The sequence identities of the cyt P450 enzymes are often very low.